Background: Although dysregulation of Hedgehog (Hh) signaling is oncogenic in several ectodermal tumors, its contributions to breast cancer have not been thoroughly explored. Our unpublished data show a significant correlation between a Hh pathway-active signature of gene expression and poor prognosis across all breast cancer subtypes. Preliminary data in a mouse model of metastatic breast cancer (MMTV-PyMT) suggest that Hh pathway activity mediated by the Gli2 transcriptional effector has opposite effects on disease progression when active in tumor epithelium as compared to tumor-associated stroma. In CK14+ tumor epithelial cells, Gli2 may enhance late-stage growth and metastasis, whereas Gli2 in the stroma may restrain initial tumor growth by promoting expression of BMP ligands. The timing, regulation, and mechanistic basis of these effects thus must be rigorously examined prior to use of Gli2-targeted therapies. These studies will illuminate the correlation between Hh pathway activity and poor prognosis, and the reduced risk of breast cancer mortality in a Chilean population subjected to high intake of arsenic, a Gli2 antagonist. Objective/Hypothesis: This proposal hypothesizes that Gli2 activity in the tumor epithelium promotes breast cancer progression while Gli2 activity in the stroma restrains tumor growth and is lost during disease progression. Simultaneously modulating these opposing Gli2 contributions using FDA-approved compounds will have therapeutic benefit. Specific Aims: 1) To determine if epithelial Gli2 is required for MMTV-PyMT tumor progression. 2) To determine whether Gli2 in the mammary stroma restrains MMTV-PyMT tumor progression. 3) To determine whether simultaneous inhibition of Gli2 in the tumor epithelium and restoration of BMP signaling provides therapeutic benefit. Study Design: Using conditional genetics to enable temporal and cell type-specific manipulation of Gli2, this study will establish the role of Gli2 in breast cancer epithelial and stromal cells using the MMTV-PyMT mouse model of metastatic breast cancer. This will enable careful characterization of the role of Gli2 at each stage of progression. Tumor growth, metastatic load, and histology will be analyzed in each experiment. The regulatory and mechanistic basis for the effects of Gli2 in the epithelium and stroma will be characterized using organoid culture and pharmacology. To circumvent the difficulty of therapeutic modulation of opposing Gli2 contributions to tumor progression in epithelium and stroma, mice will be treated with arsenic trioxide (ATO) to block Gli2 activity in epithelium, and the BMP agonist FK506 to mimic the effect of Hh pathway activity in stroma.